Making Every Nanoliter Count

Microfluidics, the science that underlies technologies for manipulating minute volumes of liquids, is the new buzzword in biology-and with good reason. Microfluidics has the potential to revolutionize the way routine molecular biology experiments are carried out. This technology is being used to miniaturize biological separation and assay techniques so that entire experiments can be accomplished within diminutive devices that are about as large as a computer chip. Picoliter-to-nanoliter volumes of solvent, sample, and reagents progress through narrow tunnels where they are mixed, separated, and analyzed by any of a variety of techniques. Microfluidic approaches offer several benefits over their standard molecular biology counterparts. The most obvious benefit is decreased sample volume (which results in decreased waste volume), but microfluidic approaches are also economical in the long run. Perhaps even more importantly, miniaturization can result in dramatically increased performance. Microfluidic technologies are highly amenable to automation and offer the potential for the massive, parallel processing of laboratory procedures. Fluid movement through the tiny vessels of microfluidics devices is fundamentally different from that through their familiar, larger counterparts. Thus, microfluidics researchers must devise ways to exploit the properties that are desirable in a given context, and to circumvent those that are not. For example, fluid flow in microfluidic channels is laminar rather than turbulent; multiple flow streams run side by side, with mixing mediated only by diffusion. This general feature is advantageous in some, but not all, cases, so some designers make adaptations such as altered micro-vessel geometry to instigate thorough intermixing when needed. They also employ different tactics to drive and control fluid flow, including electrokinetic approaches; pressure exerted by external or microfabricated "in-device" pumps; centrifugal force; and natural capillary forces resulting from attractions or repulsions between fluids and specific channel surfaces. Finally, manufacturers produce these devices from a range of materials-including silicon glass, quartz, plastic, even rubber-each with its own set of advantages and disadvantages. Microfluidic approaches have much to offer those in high-throughput (HT) environments, as well as those performing research on a smaller scale. A number of companies currently offer, or are developing, microfluidics-based devices for sample preparation and various bioanalyses. It's a Small World ACLARA Biosciences Inc. of Mountain View, Calif., combines silicon microfabrication and injection-molding techniques to manufacture plastic-based, disposable microfluidics devices. The first step is creation of a master mold, one surface of which contains a pattern of interconnected ridges. This pattern is then molded into a plastic card, and the resultant capillary networks are enclosed with film using thermal, ultrasonic, or adhesive bonding. ACLARA's Arteas™ LabCard™ devices use a 96-channel design to miniaturize biochemical assays down to nanoliter (200-600 nl) volumes, without the problems associated with evaporation. Because these devices conform to microplate design standards, laboratories can work with them using the same types of robotic equipment used for dispensing samples and reagents into microwells. ACLARA is currently developing a 32-channel "general purpose" LabCard system for highly multiplexed gene expression, proteomics, and enzyme assay applications, as well as a 96-channel HT device for these applications. The company is also devising a multi-layering method to develop chips bearing three-dimensional networks of channels. These chips will enable the design of channel "circuits," allowing one channel to jump over another when advantageous. ACLARA works with clients to adapt its system for various screening applications, and is particularly experienced in several fluorescence-based detection methodologies. Additionally, the company has developed a variety of programs with commercial customers, with applications in gene expression, DNA separation, and research clinical diagnostics. Agilent Technologies Inc. of Palo Alto, Calif., introduced the first commercially available microfluidics instrument, the Agilent 2100 Bioanalyzer, in 1999. The instrument uses disposable, glass-based LabChip® devices produced by Mountain View, Calif.-based Caliper Technology Corp. Currently available LabChip devices can separate and quantify DNA, RNA, and proteins. Chips for other applications, such as cell-based assays, are on the horizon. Electrokinetic forces drive picoliter-sized samples through the LabChip devices, where their components are resolved within interconnected networks of microchannels containing a low-viscosity, non-crosslinked polymer network for molecular sieving. The bioanalyzer's optics focus on the channel in which the separations are being carried out, and laser-induced fluorescence detects and quantifies the migrating fragments in real-time. As with the agarose gels that LabChips are designed to replace, researchers can obtain LabChips to size and quantify double stranded DNA fragments in a variety of size ranges, the broadest being 100-12,000 base pairs (bp). The newly introduced RNA 6000 Nano LabChip kit enables researchers to check the quality and determine the concentrations of total RNA and mRNA samples. The system automatically calculates the ratio of ribosomal bands in total RNA samples (5 ng) and calculates the percentage of ribosomal impurities in mRNA samples (25 ng). Sample alignment using the kit's 50-bp marker helps researchers identify and compare samples, and to troubleshoot their experiments. An optimized gel matrix and a different gel/dye mix preparation method have resulted in improved reproducibility of quantification. "Now for the first time, we can typically provide reproducibility of less than 10 percent standard deviation," explains Odilo Mueller, assay manager at Agilent. The RNA kits are designed to provide an estimation of RNA size distribution by using the bioanalyzer's software overlay feature to compare electropherograms of the sample and separately obtained sizing standards. Agilent recommends Austin, Texas-based Ambion Inc.'s RNA 6000 Ladder for this purpose (In August, the two companies entered a co-marketing agreement involving Agilent's 2100 Bioanalyzer, the RNA 6000 Nano LabChip kit, and Ambion's RNA-related reagents). Researchers can also analyze protein samples with the 2100 system, using Protein 200 Assay LabChips to obtain size and relative concentration information. The protein calibration feature in the most recent software revision (A.02.01) enables users to perform absolute protein quantification based on their own calibration standards. Thus, concentration, purity, and size information can be garnered in a single experiment. Caliper has also developed and directly markets its own LabChip-based microfluidics systems for HT environments. The Caliper 250, the company's most current HT screening platform, employs quartz sipper chips with multiple capillaries. Currently available chips contain four capillaries each, enabling up to 40,000 experiments per day, and the company plans to provide chips with 12 capillaries in the future. The company produces application-specific chips that allow researchers to perform a number of different experiments, including fluorogenic assays, mobility shift assays, fluorescence polarization assays, and cellular assays, at HT levels. Smaller is Better BioMicro Systems of Sandy, Utah, primarily works with industrial clients to develop microfluidics systems for life science research applications ranging from DNA sample processing and single nucleotide polymorphism (SNP) detection to immunoassays, toxicology testing, gene expression analysis, and proteomics. BioMicro designs microfluidic devices using hydrophobic materials. Pressure, generated by an external pump, pushes fluids through a series of passive, non-moving valves to control the location of the test sample and exposure of the sample to in situ reagents, and, therefore, the biochemical processes performed on the sample. Sunnyvale, Calif.-based Cepheid is developing fluidic cartridges to automatically carry out DNA extraction and sequence detection from a variety of sample types. The GeneXpert™ family of products, slated for commercial availability in 2003, will combine disposable cartridge-based sample preparation along with the company's I-CORE® modules for rapid DNA amplification and real-time detection. The single-use cartridges employ both microfluidic and macrofluidic technologies to extract, purify, and amplify target DNA in a series of interconnected processing chambers that contain reagents, filters, and other fluidic components required for each step. Valves are used to transfer fluids from chamber to chamber. The GeneXpert platform accepts fluidic cartridges with different internal configurations, each designed to perform a different class of assay. Thus, this technology is not restricted to DNA analyses. For example, the company is developing assays for bacterial and viral detection, detection of antibiotic resistance, cancer diagnostics by RNA detection, and SNP detection from raw blood. A bar code on the cartridge will communicate instructions to the instrument to direct fluids through the cartridge and activate mixing, lysing, amplification, detection, and other functions as required. Multiple cartridge-processing blocks can be linked to the system so that additional samples can be analyzed simultaneously. These systems will also be compact, use low power, and be suitable for applications requiring portability. The company claims that even those researchers lacking prior molecular biology experience will be able to perform DNA identification assays in only 30 minutes. "In contrast, conventional methods for DNA identification take significantly longer and are generally performed by skilled laboratory personnel," comments Kurt Petersen, Cepheid president and cofounder. As Cepheid completes development of the GeneXpert system, it has made its I-CORE technology available to the life science research market in its Smart Cycler® line of real-time PCR systems. Because Smart Cycler uses the same I-CORE modules for rapid gene amplification and detection as does the GeneXpert system, diagnostic tests originally developed to be performed using the Smart Cycler can later be migrated to GeneXpert, with the added capability of automated sample preparation. Fluidigm of South San Francisco, Calif., uses multi-layer soft lithography to fabricate rubber chips containing micro-scale valves and pumps. These on-chip valves and pumps enable the mechanical switching and pumping of minute fluid volumes. The chips can contain any of a number of tools, including mixers, sippers, dispensers, incubators, and separators, and are amenable to both high-density multiplexing and process parallelization. Fluidigm's approach allows basic or complex fluidic structures to be combined in a single, highly integrated chip. Fluidigm first separately molds the individual layers that make up a given microfluidics device. Each layer is then permanently bound to neighboring layers to yield elastomeric structures with multi-dimensional channel networks. Using this technology, Fluidigm has fabricated a chip containing 600 valves controlled by only 12 fluid control lines. The control lines run in the upper layer of the rubber chip, exerting pressure on the fluid flowing in the channels below. The company plans to work with partners to commercialize several instruments employing these chips. Its first product line will target the life sciences, with applications ranging from cell-based assays and sorting to genomic analyses, protein analyses, and biochemical assays. The company is also developing products for HT applications such as drug discovery. In July, Fluidigm and Union City, Calif.-based Axon Instruments Inc. announced plans to codevelop a microfluidics-based system for capturing and manipulating individual cells. Gyros AB of Uppsala, Sweden, develops application-specific microfluidics devices in a compact disc (CD)-like format. These discs contain large numbers of identical microstructures for the simultaneous processing of multiple samples. Gyros' microfluidics platform employs a robotic dispenser to transfer samples from microtiter plates (or other vessels) to the center of the discs' microstructures, and as the disc spins, resultant centrifugal forces push the samples towards the outside edge. As they proceed through different zones in route to the periphery, the samples undergo any of a number of different treatments such as volume definition, purification, or analysis. A laser induced fluorescence detector or other monitor type can be incorporated for data collection and analysis. According to Gyros, users can employ its basic technologies in a wide variety of applications. The company plans to release a CD for preparation of protein samples prior to identification with matrix-assisted laser desorption ionization (MALDI) mass spectrometry, and is also working on discs for immunological and cell-based assays. In May, Gyros and Stockholm, Sweden-based Affibody AB announced a collaboration to produce discs incorporating microarrays of specially designed protein-binding partners dubbed Affibodies™. Gyros has also partnered with Palo Alto, Calif.-based Genencor International to develop discs containing ready-made enzyme assays. Edmonton, Alberta-based Micralyne Inc. specializes in the design and production of micromachined glass and silicon chips. The company offers two basic microfluidics products. The MicroFluidic Toolkit (µTK) contains the electronics, optics, software, and chips to get customers up and running quickly. Users can easily reconfigure this modular system to accommodate new applications. Micralyne also sells "off-the-shelf" chips for microfluidic capillary electrophoresis, in two formats. Finally, the company will work with corporate and industrial clients to design chips for specific applications. Redmond, Wash.-based Micronics Inc. was formed in 1996 to develop and commercialize microfluidics-based products for the life sciences, drug discovery and development, diagnostics, and other applications. According to the company, the small sizes and auto-calibrating characteristics of its microfluidics devices make them especially suitable for chemical analyses and process control chemical determinations. Micronics' plastic laminate-based H-Filter® platform, named to reflect is resemblance to the letter H, enables the continuous separation of particles by size. Sample and flow streams run side by side in the center channel of the 'H' owing to laminar flow conditions; separation is effected by the differential diffusion rate of small and large molecules. Scientists can use the device for such applications as separating blood cells and plasma, extracting smaller components from sample fluids, and removing small contaminants from samples. Investigators can also use the H-Filter platform to establish stable concentration gradients between fluid layers, for example, to expose cells to various drug concentrations. In April, Micronics and Sunnyvale, Calif.-based Dionex Corp. partnered to develop devices that use H-Filter technology for real-time, online applications, which couple microfluidics sample preparation with chromatographic analysis. Micronics' T-Sensor® platform employs diffusion- based separation, and includes a self-calibration function that makes this device amenable to such applications as medical diagnostics, immunoassays, chemical reactions, and analyte detection in complex solutions. The company also offers a micro flow cytometer, dubbed the Microcytometer™ platform. Morristown, N.J.-based Honeywell is developing a wearable wristwatch-sized system that is based on this technology and that is capable of rapid measurements to provide indications of viral or bacterial infection. Finally, Micronics has developed optically resolved chemical and cellular analysis (ORCA) Microfluidics™ systems, which are custom-made, disposable microfluidic integrated circuits that enable series of assays to be performed on-chip. To familiarize customers with the company's products, Micronics offers the µFluidic™ Evaluation Kit, containing 10 different lab cards with varying circuitry. The kit is available from Rockford, Ill.-based Pierce Chemical Co. Molecular Sensing plc of Wiltshire, UK, is developing a microfluidics device called a DNA smartcard™, which is read and controlled by a device called a genedrive™. According to company literature, researchers would use these credit card-sized devices for real-time PCR applications. The DNA smartcard contains injection-molded chambers, with temperature control provided by the card's lower layer. Molecular Sensing's first commercial products are currently slated for release in 2002. Nanostream Inc. of Pasadena, Calif., develops customized microfluidics solutions for pharmaceutical and biotechnological applications. "We have developed a modular approach for rapidly prototyping microfluidic components; once optimized, the components are integrated to make devices," remarks Hugh McManus, Nanostream's vice president of sales. This prototyping approach allows for ready scale-up and mass customization with short turnaround times. Netherlands-based Micronit is another company that specializes in the design, prototyping, and development of microfluidics devices. The company produces its devices in either glass or silicon. Finally, Zurich, Switzerland-based Tecan offers the LabCD™ microfluidics platform for HT ADME (adsorption, distribution, metabolism, excretion) and toxicity assays, which currently represent a bottleneck in drug discovery. Tecan's industrial-scale platform provides all of the components needed for the company's HT ADME assay format, including software; protocols and reagents; and consumable CD-like microfluidics devices that contain microscale fluid paths, reaction chambers, and valves. The company is also developing LabCD applications for its other business areas of genomics, proteomics, and diagnostics. Creative and innovative, today's microfluidics pioneers not only explore the fundamentals of microfluidics, but also continually develop technologies for practical applications. Some microfluidics researchers are focused on harnessing different methods for driving fluids, such as electrocapillary pressure or electromagnetic forces. Others are developing alternative approaches for fluid control, including the use of electroactive surfactants or chemically controlled valves. Some envision hybrid devices with parts fabricated from different materials, such as plastic channels, glass optics, and silicon electronics. Others are experimenting with entirely different microfluidics formats, such as liquid microchannels that are created by patterning surfaces with hydrophobic and hydrophilic regions. With the plethora of ideas and possibilities swirling around the microfluidics community, the only certainty is that more innovations are coming. As these devices become more widely used, their prices will surely fall. Then, perhaps microfluidics-based instrumentation will become as commonplace in research laboratories as thermocyclers and other seminal contributions of yesteryear. Deborah A. Fitzgerald (df@sciwriters.com) is a freelance science writer in Birmingham, Ala. References 1. P. Mitchell, "Microfluidics: Downsizing large-scale biology," Nature Biotechnology, 19[8]:717-21, August 2001. 2. H. McManus, G. Dantsker, "Manipulating nano-volumes," Supplement to Chemical Specialties, s14-s18, 2001 (www.nanostream.com/pdf/010501_Supplement_to_Chemical_Specialties.pdf). 3. M. Freemantle, "Downsizing chemistry: Chemical analysis and synthesis on microchips promise a variety of potential benefits," Chemical & Engineering News, 77[8]:27-36, 1999. 4. C. Montemagno et al., online panel discussion, in "Lab-on-a-Chip: Advances in Microfluidics," by www.bio.com, Aug. 16, 2001. Supplemental Materials Microfluidics-based Systems

Making Every Nanoliter Count

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